Active enantiomer of RARγ-specific agonist

ABSTRACT

Disclosed is the (R)-enantiomer of the formula                    
     which has unexpectedly been found to possess all of the biological activity of the racemic compound disclosed in the prior art as an RARγ-specific agonist.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 60/101,747 filed Sep. 24, 1998 and U.S. provisional application Ser.No. 60/125,891 filed Mar. 24, 1999.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to preparation of the (+) or(R)-enantiomer of an RARγ-specific agonist previously described in theprior art and the discovery that all of the retinoid activity of suchagonist resided in such enantiomer.

The (R)-enantiomer of the present invention may be used in a widevariety of dermatological conditions, e.g. acne, psoriasis, eczema andphotoaging of the skin, in treatment of corneopathies in opthamology, intreatment of degenerative diseases of connective tissue, e.g. arthritis,and in the treatment of malignancies.

2. Description of the Prior Art

U.S. Pat. No. 5,624,957 discloses the racemic compound,3-fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)-2′-hydroxy)acetamidobenzoicacid (see Example 1) as an RARγ-specific retinoid with the highly usefulproperty of lacking the liver toxicity of non-elective retinoids. Thecompound is also disclosed by B. P. Klaholz, et al., Nature StructuralBiology, 5(3), pp. 199-202 (1998), as a complex with the RARγ receptorprotein. However, the compound indicated as binding to the receptor isthe (S)-enantiomer, which is the inactive form.

Although the above-described patent reference indicates that thedisclosed RARγ-specific retinoids exist in the form of the individualenantiomers as well as racemic mixtures, there is no disclosure of the(R)-enantiomer or the fact that, unexpectedly, all of the retinoidactivity of the compound of Example 1 resides in this enantiomer.

SUMMARY OF THE INVENTION

The present invention provides the compound of the formula

or a pharmaceutically acceptable salt thereof. Enantiomer IA hasretinoid-like activity and is thus useful in the treatment of skindisorders such as acne, Darier's disease, psoriasis, icthyosis, eczema,atopic dermatitis and epithelial cancers. It is also useful in thetreatment of arthritic diseases and other immunological disorders (e.g.lupus erythematosus), in promoting wound healing, in treating dry eyesyndrome and in treatment of the effects of sun damage to skin, i.e.photoaging. It is also useful in the treatment of various malignanttumors and premalignant skin lesions, e.g. actinic keratoses.

Also included in the invention is a process for preparing enantiomer IAvia chiral synthesis or separation, and pharmaceutical compositionscontaining the enantiomer IA in combination with a pharmaceuticallyacceptable carrier or diluent.

In another aspect of the invention there is provided a method fortreating a mammalian host for dermatological, rheumatic, antitumor,respiratory or opthamological conditions known to be affected byretinoid derivatives which comprises administering a therapeuticallyeffective amount of a compound of formula IA or a pharmaceuticallyacceptable salt thereof.

In yet another aspect of the invention, there is provided a method forthe prevention of spontaneous squamous cell carcinoma inimmunocompromised human transplant patients which comprises systemicallyadministering a therapeutically effective amount of a compound offormula IA.

DETAILED DESCRIPTION OF THE INVENTION

As noted above the racemic compound of the formula

is disclosed in U.S. Pat. No. 5,624,957 along with its method ofpreparation and therapeutic uses. Compound I is an RARγ-specific agonistwhich has the advantage of lacking the liver toxicity characteristic ofnon-specific retinoids.

The present inventors have discovered, surprisingly and unexpectedly,that all of the retinoid activity of compound I resides in the (+) or(R)-enantiomer IA, i.e.

The individual enantiomers of compound I may be isolated by subjectingthe allyl ester of compound I (6, below) to chiral chromatography toisolate the allyl esters of the enantiomeric acids, followed by cleavageunder mild conditions to preserve the enantiomeric purity of theproducts. The synthesis of 6 generally follows the synthesis disclosedin U.S. Pat. No. 5,624,957:

Known acid 1 (U.S. Pat. No. 5,624,957) can be reduced to the amino acid,2, using either catalytic hydrogenation or a chemical reducing agent,such as stannous chloride. Acid 2 can be converted to amino ester 3using, for example, allyl bromide. Known acid 4 is then converted to itsacid chloride and condensed with 3 to give 5, which may be reduced usingsodium borohydride to give 6.

Intermediate 6 is then subjected to chromatography on a Chiralpak ADcolumn to give 7a and 7b, which are cleaved under mild conditions (forexample, morpholine and palladium catalyst) to give the individual (R)and (S) enantiomers.

Optical purity analysis was carried out by chiral analytical HPLC,following derivatization of the free acid to the corresponding methylester under non-racemizing conditions. The determination of absoluteconfiguration was carried out by X-ray crystal analysis of 8, the(R)-Mosher ester of 7a:

The active (R) I may be enantioselectively synthesized by the followingpathway, using as a key step, the enantioselective reduction ofketoester 10 with known chiral reducing agent (R)-Alpine borane:

Known ethyl ester 9 (U.S. Pat. No. 5,624,957) is converted to allylester 10 using base hydrolysis followed by allyl bromide alkylation. 10is then enantioselectively reduced to 11 using (R)-Alpine borane. Thecrude 11 (˜94% ee) is hydrolyzed to crude 12 (94% ee), then 12 ispurified to>9 % ee via crystallization. Activation of 12 with diphosgeneand condensation with amino ester 3 gives 7a, whose ester group iscleaved to give the final product, (R) I (ee>99%).

Compound IA may be converted with bases to pharmaceutically acceptablesalts thereof by methods known in the art. Examples of suitable saltsare ammonium and alkali metal salts, especially of sodium, potassium andlithium, and alkaline earth metal salts, especially calcium andmagnesium, as well as salts with suitable bases such as with loweralkylamines, e.g. methylamine, ethylamine or cyclohexylamine, or withsubstituted lower alkylamines such as diethanolamine or triethanolamineand with piperidine or morpholine.

As noted above, the compound of the present invention has retinoid-likeactivity and can, therefore, be used for the treatment ofdermatological, rheumatic, antitumor, respiratory and opthalmologicalconditions know to be affected by retinoid derivatives. For example, thecompound may be used for the treatment of:

dermatological conditions linked to a disorder of keratinisationinvolving differentiation and proliferation, e.g. in treating acnevulgaris, comedonic or polymorphic acne, nodulocystic acne, acneconglobata, senile acne and secondary acnes such as solar, drug andoccupational acne;

for treating other types of keratinisation disorders such as ichthyoses,ichthyosiform states, Darier's disease, palmoplantar keratoderma,leucoplakia and leucoplakiform states, and lichenplanus;

for treating dermatolgical conditions linked to a keratinisationdisorder with an inflammatory and/or immunoallergic component, e.g. allforms of psoriasis, whether cutaneous, mucosal and ungual, and psoriaticrheumatism, or alternatively, cutaneous atopy, such as eczema, orrepiratory atopy;

for treating dermal or epidermal proliferations, whether benign ormalignant, including those of vital origin, such as common warts, flatwarts and epidermodysplasia verruciformis;

for treatment of other dermatological disorders such as vesiculardematoses and collagen diseases;

for treatment of certain opthalmological disorders: in particularcorneopathies;

for prophylaxis or treatment of skin aging, both light induced(photoaging) and that occurring with the passage of time;

for preventing or treating the stigmata of epidermal and/or dermalatrophy induced by local or systemic corticosteroids, or any other formof cutaneous atrophy;

for treatment of malignant tumors;

for treatment of premalignant skin lesions such as actinic keratosis;

for rheumatic illnesses, especially those of an inflammatory ordegenerative kind which attack the joints, muscles, tendons and otherparts of the motor apparatus, e.g. rheumatic arthritis;

for promoting cicatrisation; and

for combating disorders of sebaceous function, such as seborrhea of acneor simple seborrhea.

Skin cancers, especially squamous cell carcinomas, are the most frequentmalignancies in immunocompromised patients, e.g. organ transplantrecipients. Systemic retinoids such as isotretinoin have been studiedfor prevention of spontaneous squamous cell carcinomas, but adverseside-effects on long-term use such as liver toxicity limit theirusefulness. Compound IA, however, lacking the liver toxicity ofnon-specific retinoids, is especially useful for this indication. Thusthe present invention includes the method of preventing spontaneoussquamous cell carcinomas in immunocompromised human transplant patientswhich comprises systemically administering a therapeutically effectiveamount of a compound of formula IA.

The compounds of the present invention can be administered orally,parenterally or topically, depending on such considerations as thecondition to be treated, need for site-specific treatment, quantity ofdrug to be administered, and similar considerations. They are generallyused as pharmaceutical compositions with one or more suitablepharmaceutical carriers or diluents conventionally used inpharmaceutical technology.

In the treatment of dermatological conditions, it will generally bepreferred to administer the compounds topically, although in certaincases such as treatment of severe acne or psoriasis, oral formulationwill be employed. For other indications, parenteral, oral or topicaladministration may be preferred. The pharmaceutical compositions may bein solid form such as capsules, tablets, powders, gels, salves,ointments, etc. or in liquid form such as solutions, suspensions oremulsions. For parenteral administration, the drug may be prepared inunit dose form in ampules or in multidose containers and may containadditives such as suspending, stabilizing and dispersing agents. Theparenteral compositions may be in ready-to-use form or in powder formfor reconstitution at the time of delivery with a suitable vehicle suchas sterile water. Illustrative examples of suitable pharmaceuticalformulations are disclosed, for example, in U.K. 2,164,938A.

The compound of the present invention may be administered alone or inadmixture with other medicaments, e.g. agents for treating skin dryness,providing protection against photoaging, preventing infection, reducingirritation and inflammation, and the like.

The dosages and dosage regimen in which the compound of the presentinvention are administered will vary according to the compound, dosageform, mode of administration, the condition being treated and theparticulars of the patient being treated. Accordingly, optimaltherapeutic concentrations will be best determined at the time ofadministration by conventional dosage determination procedures. Ingeneral, however, the compounds may be administered in amounts of about0.05 mg to about 5 mg daily per kg of body weight in one or more doses.

Isotretinoin (Accutane®) and etretinate (Tegison®) are used clinicallyto treat severe recalcitrant cystic acne and severe recalcitrantpsoriasis, including the erythrodermica and generalized pustular types,respectively. Their mode of use is amply illustrated in the Physician'sDesk Reference, 47th Edition (1993), published by Medical EconomicsData. The compounds of the present invention may be administered in asimilar fashion to isotretinoin and etretinate according to theseguidelines. For treatment of other indications, such as tumors, thecompounds of the present invention may be administered to mammals,including humans, in a similar manner to retinoid compounds in theliterature which have been shown to be active for such indications.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The specific example which follows illustrates the synthesis of theenantiomer of the present invention.

Definitions for some of the abbreviations used below are as follows:

DMSO dimethylsulfoxide CDCl₃ deuterated chloroform EtOH ethyl alcoholDMF dimethylformamide ee enantiomeric excess EtOAc ethyl acetate Et₃Ntriethylamine IPA isopropyl alcohol DCC dicyclohexylcarbodiimide Phphenyl THF tetrahydrofuran TMS trimethylsilyl

A. 4-Amino-3-fluorobenzoic acid, 2-propenyl ester, 3

10% Pd/C (1.98 g) was added to a 450 mL hydrogenation flask, and theflask was flushed with N₂. A solution of nitro benzoic acid 1 (21.9 g,118.3 mmol) in 140 mL ethanol and acetic acid (1 mL) were added to theflask. Hydrogenation was conducted at 15 psi in a carefully controlledmanner, wherein after the pressure of H₂ drops to zero, the shaking ofthe flask was continued for a couple of minutes before re-pressurizing.After the hydrogen uptake ceased, the pressure was raised to 40 psi, andcontinued shaking for an additional 30 minutes to insure completion ofthe reduction. The catalyst was filtered through Celite, and solvent wasremoved in vacuo to afford amino benzoic acid 2 as an off-white solid.

The crude acid 2 was dissolved in DMF (140.0 mL), treated with K₂CO₃(16.0 g, 115.8 mmol) and allyl bromide (11.8 mL, 132.3 mmol), andvigorously stirred at room temperature for 24 hours. The crude reactionmixture was treated with 1N HCl (120 mL) carefully. It was then dilutedwith water (70 mL), and extracted with CH₂Cl₂ (400 mL). The organiclayer was washed with water (70 mL, 5×) and brine. It was dried withMgSO₄, filtered and evaporated in vacuo to afford a crude oil. The crudeoil was purified with flash chromatography (silica gel; 10-20%EtOAc/hexanes) to afford aniline 3 as a faint yellow solid (20.4 g,combined yield).

3: Mp 53.5-55.5° C. IR (KBr): 3418, 3337, 3215,1708, 1639, 1609, 1582,1522. ¹H NMR (CDCl₃, δ=7.28): 7.73-7.68 (m, 2H, C-2H & C-6H), 6.77 (appt, J═8.6, 1H, C-5H), 6.03 (m, 1 H, OCH₂CH), 5.40 (dm, J=16.4, 1H, ═CH₂trans), 5.28 (dm, J=10.4, 1H, ═CH₂ cis), 4.79 (dm, J=5.6, 2H, OCH₂),4.21 (br s, 2H, NH₂). LRMS: (ESI) m/z (M−H)⁻=194.3.

Anal. Calcd. for C₁₀H₁₀FNO₂: C, 61.53; H, 5.16; N 7.18. Found: C, 61.60;H, 5.18; N, 7.16.

B.3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-oxo)acetamidobenzoic acid, 2-propenyl ester, 5

Et₃N (16.0 mL, 114.8 mmol) was added over five minutes to a cooled (0°C.) CH₂Cl₂ (100.0 mL) solution of acid 4 (10.15 g, 39.00 mmol) and SOCl₂(8.0 mL, 109.7 mmol). The cooling bath was removed 10 minutes later, andstirring was continued at room temperature for additional 1 hour and 50minutes. The reaction mixture was diluted with CH₂Cl₂, and washedquickly with water and dried over MgSO₄. It was filtered, and solventwas removed in vacuo to afford a dark brown viscous oil, which wassubmitted to the coupling step without any purification.

Et₃N (8.0 mL, 57.4 mmol) was added drop-wise over a few minutes to anEtOAc (110.0 mL) solution of the allyl benzoate 3 (7.30 g, 37.6 mmol)and the acid chloride prepared above. It was then stirred overnight. Themixture was diluted with EtOAc, and washed with water and brine. Theorganic layer was dried with MgSO₄, filtered and evaporated in vacuo.Flash chromatography (the oil was loaded directly to silica gel; 5-7%EtOAc/hexanes) afforded ketoamide 5 as a red-brown viscous oil whicheventually solidified to a dense solid. It weighed 11.38 g (a combinedyield of 67%).

5: IR (KBr) 3352, 2959, 2922, 1719, 1705, 1670, 1618, 1599, 1528. ¹H NMR(CDCl₃, δ=7.28) 9.42 (br s, 1 H, NH), 8.63 (app t, J=8.1, 1H, NHCCH),8.43 (d, J=1.8, 1H, HCCCO), 8.18 (dd, J=8.4, 1.8, 1H, HCCCO), 7.96 (d,J=8.7, 1H, FCCH), 7.87 (dd, J=11.2, 1.8, 1H, NCCHCH), 7.47(d, J=8.4, 1H, CHCHCCO), 6.05 (m, 1H, OCH₂CH), 5.44(dm, J=17.2, 1 H, ═CH₂ trans),5.34 (dm, J =10.4, 1H, ═CH₂ cis), 4.85 (dt, J=5.7, 1.2, 2H, OCH₂), 1.75(s, 4H, CH₂CH₂), 1.37(s, 6H, CH₃/CH₃), 1.34 (s, 6H, CH₃/CH₃). LRMS:(ESI) m/z (M−H)⁻=436.4.

C.3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-hydroxy)acetamidobenzoic acid, 1(2-propenyl) ester, 6

NaBH₄ (16.5 mg, 0.436 mmol) was added in one portion to an allyl alcohol(11.0 mL) solution of ketoamide 5 (450.0 mg, 1.029 mmol). The reactionmixture was stirred vigorously for 10 minutes, then quenched with 2drops of concentrated HCl, and partitioned between EtOAc (150 mL) anddilute NaHCO₃ solution (i.e., 5 mL saturated NaHCO₃ solution+45 mLwater). The water layer was back extracted with EtOAc (50 mL). Thecombined organic phase was washed with brine and dried with MgSO₄. Itwas then filtered and evaporated. Flash chromatography of the crudematerial (sample was loaded as a silica gel mesh; 20-25% EtOAc/hexanes)afforded alcohol 6 as a colorless oil which solidified slowly at roomtemperature. It weighed 412 mg (yield=91.1%).

6: IR (KBr) 3600-3150 (br), 3366, 2959, 1719, 1692, 1620, 1593, 1532. ¹HNMR (CDCl₃, δ=7.28) 8.75 (br s,1H, NH), 8.51 (app t, J=8.2, 1H, FCCCH),7.88 (d, J=8.7, 1H, FCCH), 7.81 (dd, J=11.4, 1.8, 1H, NCCHCH), 7.42 (d,J=1.9, 1H, CCHCCO), 7.37 (d, J=8.2, 1H, CHCHCCHOH), 7.25 (dd, J=8.2,1.9, 1H, CHCHCCHOH), 6.04 (m,1H, OCH₂CH), 5.42 (dm, J=17.2, 1H, ═CH₂trans), 5.32 (dm, J=11.7, 1H, O═CH₂ cis), 5.25 (d, J=3.0, 1H, CHOH),4.83 (dm, J=5.7, 2H, OCH₂), 3.05 (d, J=3.0, 1H, OH), 1.70 (s, 4H,CH₂CH₂), 1.33 (s, 3H, CH₃), 1.30 (s, 3H, CH₃), 1.29 (s, 6H, CH₃/CH₃).LRMS: (ESI) m/z (M−H)⁻=438.3.

D. (R) and(S)-3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-hydroxy)acetamido benzoic acid, 2-propenyl ester, 7a and 7b

The resolution of alcohol 6 was effected on Waters HPLC system with a4000 series system-control and a 490E series detector according toconditions described below. The elution pattern was monitored at fourwavelengths (210, 254, 280 and 300 nm) where similar absorption profileswere observed.

Column: Chiralpak AD (5 cm×50 cm) pre-equilibrated with 60:40hexanes/IPA for 0.5 hour.

Sample Loading: 3.0 g of alcohol 6 was added to 40 mL of 1:1hexanes/IPA. The mixture was sonicated with moderate heating (˜40° C.)until total dissolution was effected. The solution was removed from thesonicating bath and allowed to cool to room temperature. It was thenloaded directly onto the column at 10 mL/minutes.

Elution: Elution of the column was carried out with a 60:40 hexanes/IPAsolution at 50 mL/minutes. Sample collection was carried out manually inthree fractions: the first enantiomer (7a) came out between 15-25minutes; the intermediate fraction was discarded, and the last fractionwas the second enantiomer (7b) which eluted between˜40-65 minutes.

Removal of solvent in vacuo afforded a viscous oil, in both cases.

Analysis of Optical Purity: Both fractions were determined to beoptically pure when analyzed according to the following conditions:

Instrument: HP 1090 Liquid Chromatography with DAD

Column: Chiracel AD, 0.46 cm×25 cm

Mobile Phase: 80/20 (hexanes/IPA)

Flow Rate: 1.5 mL/min

Detection: UV absorption @ 210 nm

Sample was prepared in 1:1 hexanes/IPA

Elution time: alcohol 7a (2.86 min) and alcohol 7b (10.92 min)

E. (R)3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-((R)-α-methoxy-α-trifluoromethylphenylacetoxy))acetamido benzoic acid, 2-propenyl ester, 8

DCC (115.0 mg, 0.557 mmol) was added in one portion to a CH₂CI₂ (3.0 mL)solution of (R)-Mosher acid (127.0 mg, 0.542 mmol), alcohol 7a (197.0mg, 0.449 mmol) and DMAP (5.2 mg, 0.043 mmol). The reaction was stirredfor a total of 4 hours and filtered through a cotton plug to remove theurea byproduct. The solvent was removed in vacuo and the resulting oilwas submitted to flash chromatography (silica gel; 10-15% EtOAc/hexanes)to afford Mosher ester 8 as a white foam (278 mg, 94%). X-ray qualitycrystals were grown in EtOH (˜2 mL) with a few drops of water, at roomtemperature. The absolute structure of the benzylic position of 7A wasdetermined to be (R).

F. (R)3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-hydroxy)acetamidobenzoic acid, (R)-IA

Pd(Ph₃P)₄ (216.0 mg, 0.187 mmol) was added in one portion to a THF (50.0mL) solution of allyl ester 7a (3.00 g, 6.826 mmol) and morpholine (5.0mL, 57.34 mmol). The solution was stirred for 40 minutes, diluted withEtOAc (150 mL), and washed with 1N HCl (40 mL, 2×) and brine. Theorganic layer was dried with MgSO₄, filtered and evaporated in vacuo toafford a solid. The sample was purified with flash chromatography (shortcolumn; sample was loaded as a silica gel mesh; 75 hexanes: 24 EtOAc:0.5 of 90% HCO₂H: 0.5 MeOH) to afford acid (R) I as a white solidweighing 2.50 g (91.6%). Recrystallization: about 8 mL of EtOAc wasadded to the solid, and the mixture was heated until total dissolutionwas effected. Upon addition of hexanes (65 mL) to the solution, whitecrystals started forming immediately. Half hour later, the solid wasfiltered and washed with 20% EtOAc/hexanes. Exposure of the sample tohigh vacuum afforded 1.824 g of acid.

(R) I: MP 193.0-195.5° C.

Anal. Calcd. for C₂₃H₂₆FNO₄: C, 69.16; H, 6.56; N, 3.51. Found: C,69.07; H, 6.57; N, 3.31.

(S) I: MP 194.5-197° C.

Anal. Calcd. for C₂₃H₂₆FNO₄: C, 69.16; H, 6.56; N, 3.51. Found: C,69.01; H, 6.51; N, 3.28.

Specific Rotation [MeOH, 25°] Wave- length Allyl ester 7a Allyl ester 7bAcid (R) IA Acid (S) IB 589 nm +2.577 −2.476 +1.720 −1.630 578 nm +3.319−3.461 +2.271 −2.390 546 nm +5.072 −5.399 +3.875 −4.235 436 nm +22.307 −23.525 +21.008 −21.855 365 nm — −81.153 +77.404 −78.629

Optical purity analysis of the methyl esters: Acid IA (31.4 mg, 0.0786mmol) was dissolved in 3.0 mL of C₆H₆/MeOH (7:2) mixture, and treatedwith TMS-diazomethane (200 μL of 2.0 M in hexanes, 0.40 mmol). After thereaction mixture was stirred for 11 minutes, the excess reagent wasquenched with 2 drops of acetic acid. Most of the volatile component wasremoved in vacuo, and the crude material was directly submitted to flashchromatography (silica gel; 15% EtOAc/hexanes) to afford the methylester of IA as a colorless oil. HPLC analysis of the ester on an ADcolumn indicated that it was optically pure. (Elution time: (R)-methylester =3.00 min; (S)-methyl ester =11.17 min)

G.2-Oxo-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-napth-2′-yl)aceticacid, 2-propenyl ester, 10

A KOH solution (150.0 mL of 1.76 M, 264.0 mmol) was added to an EtOH(350 mL) solution of ethyl ketoester 9 (50.3 g, 174.4 mmol). After athorough mixing, the solution was allowed to stand at room temperaturefor 30 minutes. It was diluted with water (500 mL) and acidified with 5%HCl to pH 3-4. The aqueous layer was extracted with EtOAc (1.0 L and 250mL). The combined organic phase was washed with brine, dried with MgSO₄,filtered and evaporated in vacuo. The resulting crude oil was exposed tohigh vacuum over night to afford acid 4 as a yellow solid.

K₂CO₃ (24.0 g, 173.6 mmol) and allyl bromide (18.0 mL, 208.0 mmol) wereadded to a DMF (200 mL) solution of the crude acid. The allylation wascomplete with in 150 minutes. The reaction mixture was slowly treatedwith 1 N HCl (170 mL), with vigorous stirring. The resulting mixture wasdiluted with water (100 mL), and extracted with CH₂Cl₂ (500 and 100 mL).The combined organic phase was washed with water (100 mL, 5×), andbrine, dried with MgSO4, filtered and evaporated in vacuo. The crude oilwas purified with flash chromatography (silica gel; 10% EtOAc/hexanes)to afford ester 10 as a dense solid (50.0 g, 95% combined yield).

10: IR (KBr): 2963, 2930, 2869, 1736, 1682, 1600. ¹H NMR (CDCl₃,δ=7.28): 8.00 (d, J=1.9, 1H, C-1H), 7.73 (dd, J=8.3, 1.9, 1H, C-3H),7.45 (d, J=8.3, 1H, C-4H), 6.05 (m, 1H, CH═CH₂), 5.47 (dm, J=17.2, 1H,═CH₂ trans), 5.37 (dm, J=10.5, 1H, ═CH₂ cis), 4.89 (dm, J=5.9, 1H,OCH₂), 1.73 (app s, 4H, CH₂CH₂), 1.324 (s, 6H, CH3/CH₃), 1.320 (s, 6H,CH3/CH3). LRMS: (ESI) m/z (M−H)⁻=259.4.

Anal. Calcd. for C₁₉H_(24O) ₃: C, 75.97; H, 8.05. Found: C, 75.92; H,8.21.

H.(R)-2-Hydroxy-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-napth-2′-yl)acetic acid, 1(2-propenyl)ester, 11

Ketoester 10 (33.60 g, 111.85 mmol) was ground up with a mortar andpestle and transferred into a 1L round-bottomed flask equipped with amagnetic stirrer. The flask was then flushed with nitrogen.(R)-Alpine-Borane (57.0 mL, 202.7 mmol; 97% neat liquid) was transferredto the reaction flask via syringe. The mixture was vigorously stirred atroom temperature for a total of 64 hours.

To quench the excess Alpine-Borane, the mixture was cooled to 15° C. andtreated with acetaldehyde (16.8 mL, 300.5 mmol). A few minutes later,the cooling bath was removed and the reaction mixture was stirred atroom temperature for 45 minutes. The reaction mixture was then flushedwith N₂ while being heated with a water bath of 45° C. which was allowedto cool down to room temperature over the next 3.75 hours.

Ether (200 mL) was added to the reaction mixture. The resulting solutionwas cooled to ˜10° C. and treated with ethanol amine (14.5 mL, 240.2mmol) drop-wise over a few minutes. The cooling bath was removed and themixture was stirred at room temperature for an additional 30 minutes.

The mixture was filtered, and the white precipitate was washed withhexanes (140 mL). The filtrate was diluted with EtOAc (200 mL) andwashed with dilute HCl solution (prepared from 200 mL water and 5 mL of5% HCl). The organic layer was washed with brine, dried with MgSO₄,filtered and evaporated in vacuo to afford a semi-viscous oil.

The crude material was directly submitted to flash chromatography(silica gel; 7.5-20% EtOAc/hexanes) to afford impure hydroxy ester 11(33.8 g) as a colorless oil.

I.(R)-2-Hydroxy-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-napth-2′-yl)acetic acid, 12

Morpholine (42.0 mL, 481.6 mmol), followed by Pd(Ph₃P)₄ (1.00 g, 0.865mmol) were added to a THF (420 mL) solution of 33.8 g impure hydroxyester 11. The flask was flushed with N₂ for a few minutes, and thereaction mixture was stirred for an additional 50 minutes. It was thendiluted with EtOAc (700 mL) and washed with 1 N HCl solution (230 mL,2×), and brine. The organic layer was dried with MgSO₄, filtered andevaporated in vacuo to afford an oil containing yellow precipitate.

The crude material was directly submitted to silica gel flashchromatography. The column was first flushed with 25% EtOAc/hexanes,then eluted first with 75:25:0.5:0.5 hexanes/ EtOAc/MeOH/90% formicacid, followed by 75:25:0.5:0.5 EtOAc/ hexanes/MeOH/90% formic acid. Twofractions of the desired material were collected: a slightly impurefraction 12 as a white foam (2.46 g), and a pure hydroxy acid 12 as anoff-white dense solid (19.5 g; a two step combined yield of >67%).

Optical purity analysis of acid 12 with HPLC: Acid 12 was converted toits methyl ester according to the procedure described for IA, with theexception that 10% EtOAc/hexanes was employed in the flashchromatography purification. Analysis of the resulting colorless oilaccording to the conditions noted below gave ˜94% ee.

Chiral HPLC Analysis

Instrument: HP 1090 Liquid Chromatography with DAD

Column: Chiracel OD, 0.46 cm×25 cm

Mobile Phase: 95:5 hexanes/IPA

Flow Rate: 1.0 mL/min

Detection: UV absorption @ 210 nm

Sample was prepared in 1:1 hexanes/IPA

Elution time: 6.92 min (S-hydroxy methyl ester); 8.73 min (R-hydroxymethyl ester)

Optical purity enhancement of hydroxy acid 12 via recrystallization:Several batches of hydroxy acid 12 with an ee in the range of 93-94%,and a total weight of 57.0 g were mixed and ground up with a mortar andpestle. The material was divided into two equal batches, and each wasdissolved in 140 mL EtOAc at room temperature, treated with 280 mLhexanes, and then stored in a refrigerator for 21 hours. The precipitatewas filtered and washed with 100 mL of 10% EtOAc/hexanes and air dried.The two sets afforded a total of 23.3 g of white fluffy solid. ChiralHPLC analysis of its methyl ester derivative indicated an ee >99.5%.

A second crop gave 7.3 g; chiral HPLC analysis indicated an ee >99.5%),and a third crop gave 8.3 g with an ee of 99.4%.

12: MP 145.0-147.5° C. IR (KBr): 3433, 3389, 2959, 2922, 2857, 1739,1728. ¹H NMR (CDCl₃, d=7.28): 7.38 (d, J=1.9, 1H, C-1 H), 7.33 (d,J=8.2, 1H, C-4H), 7.20 (dd, J=8.2, 1.9, 1H, C-3H), 5.23 (s, 1H, CHOH),1.70 (app s, 4H, CH₂CH₂),1.30 (s, 3H, CH₃),1.29 (s, 3H, CH₃), 1.28 (s,6H, CH₃/CH₃). LRMS (ESI) m/z (M−H)⁻=261.4.

Anal. Calcd. for C₁₆H₂₂O₃: C, 73.25; H, 8.45 Found: C, 73.47; H, 8.34.

[α]²⁵D=−100.85° (c, 1.016, MeOH).

J.(R)-3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-hydroxy)acetamidobenzoic acid, 1(2-propenyl) ester, 7a

A THF (50.0 mL) solution of trichloromethyl chloroformate (12.0 mL, 99.5mmol) was added dropwise over 4 minutes to a THF (200.0 mL) solution ofhydroxy acid 12 (26.2 g, 99.9 mmol). The solution was heated with an oilbath (63-65° C.) for 5 hours and 10 minutes. The oil bath was replacedwith a water bath of 35° C., and the reaction mixture was flushed withN₂ for 1.5 hours. Most of the left-over volatile component of thereaction mixture was removed in vacuo, and the resulting crude oil wasexposed to high vacuum for 1 hour with intermittent N₂ flushing. Thecrude was diluted with CH₂Cl₂ and evaporated. The resulting oil wasexposed to high vacuum for 45 minutes with intermittent N₂ flushing togive 12a.

Aniline 3 (17.7g, 90.7 mmol) was added to a CH₂Cl₂ (195.0 mL) solutionof crude dioxolanedione (12a) over a few minutes. The reaction mixturewas stirred for a total of 16 hours. It was diluted with CH₂Cl₂ (450 mL)and washed with water (220 mL). The CH₂Cl₂ layer was dried with MgSO₄,filtered and evaporated in vacuo. The crude material was then purifiedwith repeated flash chromatography (silica gel; CH₂Cl₂ elution to afforda mixture of aniline 12 and alcohol 7a, followed by 20% EtOAc elution toafford clean alcohol 7a). The material from the CH₂Cl₂ elution wasresubmitted to the same condition to afford more clean coupled material.A white foam (33.7 g, 77% yield) was obtained. ee >99.5%.

7a: IR (KBr): 3442 (br), 3374, 2961, 2928, 2861, 1724, 1699, 1620, 1594,1527. ¹H NMR (CDCl₃, δ=7.28): 8.77 (br d, J=2.8, 1H, NH), 8.51 (app t,J=8.1, FCCCH), 7.87 (d, J=9.2, 1H, FCCH), 7.80 (dd, J=11.4, 1.8, 1H,CHCHCO₂), 7.42 (d, J=1.9, 1H, CCHCCOH, 7.36 (d, J=8.2, CCHCHCCOH), 7.25(dd, J=8.2, 1.9, CCHCHCCOH), 6.02 (m, 1H, OCH₂CH), 5.42 (dm, J=17.2, 1H,═CH₂ trans), 5.32 (dm, J=10.4, 1H, ═CH₂ cis), 5.24 (d, J=2.5, 1H, CHOH),4.83 (dm, J=5.7, 2H, OCH₂), 3.11 (d, J=2.5, 1H, CHOH), 1.70 (s, 4H,CH₂CH₂),1.33 (s, 3H, CH₃), 1.30 (s, 3H, CH₃),1.29 (s, 6H, CH₃/CH₃). LRMS(ESI) m/z (M−H)⁻⁼438.5.

Anal. Calcd. for C₂₆H₃₀FNO₄: C, 71.05; H, 6.88; N, 3.19. Found: C,70.79; H, 6.87; N, 3.14.

[α]^(D) ₂₅=+2.50 (c, 1.898, MeOH).

K.(R)-3-Fluoro-4(2′(5″,6″,7″,8″-tetrahydro-5″,5″,8″,8″-tetramethyl-2″-naphthyl)2′-hydroxy)acetamidobenzoic acid, (R) IA

Pd(Ph₃P)₄ (0.55 g, 0.476 mmol) was added to a THF (190.0 mL) solution ofallyl benzoate 7a (20.55 g, 46.76 mmol) and morpholine (29.0 mL, 332.5mmol). The reaction mixture was stirred for 20 minutes. It was dilutedwith EtOAc (300.0 mL) and washed with 1N HCl (170.0 mL, 2×) and brine,and dried with MgSO₄. The mixture was filtered and evaporated in vacuo.Purification with flash chromatography (sample was loaded as a silicagel mesh; 75:25:0.5:0.5 hexanes/EtOAc/MeOH/90% formic acid→60:40:0.5:0.5EtOAc/hexanes/MeOH/90% formic acid) afforded acid (R) IA as an off-whitesolid. The acid was dissolved in EtOAc (58 mL) with heating; then wereadded hot hexanes (470 mL) during one minute. The solution was cooledand the precipitate was filtered and washed with 100 mL of 20%EtOAc/hexanes. Acid (R) IA was retrieved as a white shiny solid (16.4 g,87.8% yield).

(R) IA: Mp=194.5-199.0° C. IR (KBr) 3565, 3421, 3396, 3068, 2957, 2924,2904, 2856, 1721, 1685, 1676, 1618, 1592, 1526. ¹H NMR (DMSO, δ=2.51)13.12(s, CO₂H), 9.79 (d, J=1.5, NH), 8.10 (app t, J=8.3, NHCCH),7.78-7.72 (m, 2H, FCCHCCH), 7.46 (d, J=1.5, CCHC), 7.30 (d, J=8.1, 1H,CHCHCCOH), 7.21 (dd, J=8.1, 1.5, 1H, CHCHCCOH), 6.58 (d, J=4.5, 1H, OH),5.16 (d, J=4.5, CHOH), 1.63 (s, 4H, CH₂CH₂),1.25 (s, 3H, CH₃),1.24 (s,3H, CH₃), 1.22 (s, 6H, CH₃/CH₃). LRMS (ESI) m/z (M−H)⁻⁼398.5.

Anal. Calcd. for C₂₃H₂₆FNO₄: C, 69.19; H, 6.56; N, 3.51. Found: C,69.23; H, 6.37; N, 3.44.

[α]^(D) ₂₅=+1.13 (c, 2.113, MeOH). Chiral HPLC analysis of the methylester derivative: ee >99.5%.

Biology

The transactivation assay measures the ability of a retinoid to activatea reporter gene in the presence of one of the retinoic acid receptorsubtypes (α, β, or γ). The details of the receptor-based transactivationassay are disclosed in the literature, e.g. see Nature 1988, 332,850-853. In the retinoid transactivation assay, HeLa cells areco-transfected with DNA encoding RAR α,βor γ, and an RAR responsive CAT(chloramphenicol acetyl transferase) reporter gene. Retinoid efficacy ismeasured by the concentration of induced CAT gene product as determinedby ELISA assay. The dosage at which CAT level is ½ the maximum level istermed the EC₅₀. The mean EC₅₀ value for each of the receptors iscalculated using a computer generated induced-fit program. The followingtable reports the EC₅₀ values for both enantiomers (in nM):

Transactivation ED₅₀ (% max) Compound RAR-α RAR-β RAR-γ (R) I NA 300(86) 20 (98) Racemate NA 400 (31) 30 (80) (S) I NA NA NA

All the activity resides in the R enantiomer.

Non-receptor-selective retinoids have been shown to prevent theconversion of papillomas to malignant tumors in the two-stage system ofmouse skin carcinogenesis, where DMBA (7,12-dimethyl-benzanthracene) isused as the initiator and 12-tetradecanoyl-phorbol-13 acetate (TPA) isused as the promoter. The model and results are described in, forexample, L. C. Chen, et al.; Cancer Letters, 78, pp. 63-7 (1994); D. R.Shalinsky, et al., Proc. Ann. Meet. Am. Assoc. Cancer Res., 35, p. A-831(1994); and L. C. Chen, et al.; Carcinogenesis, 15, pp. 2383-6 (1994).They have also been shown to be of benefit in human organ transplantpatients, who are at increased risk of developing malignant skin tumorsdue to the required immunosuppressive therapy. Clinical studies aredescribed in S. Euvrard, et al., BioDrugs, 8, pp. 176-84 (1997); J. N.B. Bavinck, et al., J. Clin. Oncol., 13, pp.1933-8 (1995); and G. E.Gibson, et al., J. Eur.Acad. Dermatol. Venereol., 10, pp. 42-7 (1998).The present inventors have now discovered that the active, enantiomer IApossesses superior activity in this model.

The model used was essentially the same as described in the referencesabove. Compound IA and 13-cis-retinoic acid (control) were given toseveral groups of mice by intraperitoneal injection at the start of theTPA promotion phase, and the number and size of papillomas weremonitored for 10 weeks. Compound IA at doses of 15 mg/kg or highersignficiantly reduced both the number and size of papillomas, while13-cis-retinoic acid at 50 mg/kg was inactive under these conditions.The results of the study are summarized in the table below:

Tumor Incidence Tumor Burden, mm³ (% of mice (mean tumor size Treatmentwith tumors) per mouse) IA (5 mg/kg) 70 7.0 IA (15 mg/kg) 45 1.2* IA (30mg/kg) 15 0.8* 13-cis-retinoic acid 80 6.8 Vehicle 90 6.6 Untreated 906.8 *statistically significant difference (p < 0.05) vs. vehicle oruntreated

We claim:
 1. A method for the prevention of spontaneous squamous cellcarcinoma in immunocompromised human transplant patients which comprisessystemically administering a therapeutically effective amount of acompound of the formula

or a pharmaceutically acceptable salt thereof.